10
Cell-mediated cytotoxicity
■ Cytotoxic T cells recognize antigen presented on
MHC molecules. Most cytotoxic T cells are CD8+ and
recognize antigenic peptides presented on MHC
class I.
■ Natural killer (NK) cells react against cells which do not
express MHC class I. They can interact with these cells
using a variety of receptors.
■ NK cells express two major classes of inhibitory
receptors for MHC molecules. These are lectin-like
receptors of the CD94 family and immunoglobulin
superfamily molecules (KIRs).
■ Cytotoxicity is mediated by combinations of direct
The last two chapters dealt with the types of immune
reaction which are controlled by T helper (TH) cells,
namely antibody responses directed by TH2 cells and cellmediated immunity mediated by macrophages and TH1
cells. This chapter is concerned with cytotoxicity, the ways
in which leucocytes recognize and destroy other cells.
Cell-mediated cytotoxicity is an essential defence against
intracellular pathogens, including viruses, some bacteria
and parasites. Tumour cells, eukaryotic pathogens and
even cells of the body, may also become the target of
cytotoxic cells. Addtionally, the process is important in the
destruction of allogeneic tissue grafts. Several types of cell
can execute this activity including T-cytotoxic (TC) cells,
NK cells and sometimes myeloid cells. The mechanisms
of recognition and killing used by the lymphoid cells are
quite distinct from those of the myeloid cells, and will be
considered first.
Cytotoxic T cells and NK cells are
complementary elements in the immune
defence against virally infected cells
Cytotoxic T cells and NK cells recognize their targets in
different ways (Fig. 10.1).
Cytotoxic T cells recognize specific antigens (e.g. viral
peptides on infected cells) presented by MHC molecules.
Most T C cells are CD8 + and recognize antigen presented
on MHC class I, but about 10% of MHC-restricted cytotoxic T cells are CD4+ and recognize antigen presented on
class II molecules.
NK cells recognize cells which fail to express MHC class
I molecules. These cells also use a variety of receptors
to recognize their targets positively. For example, they can
bind to antibody already attached to antigen on a target
cell, using their Fc receptors (CD16): this is known as
antibody-dependent cell-mediated cytotoxicity (ADCC),
or killer cell (K cell) activity.
The most important role of TC cells is the elimination
of cells infected with virus (see Chapter 14). Nearly all
nucleated cells express MHC class I molecules and if they
become infected they can therefore present antigen to
CD8+ TC cells. The detailed mechanisms of antigen presentation to Tc cells are discussed in Chapters 5 and 6 and
cell–cell interactions, cytokines and the release of
granule proteins. Fas ligand and tumour necrosis
factors can signal apoptosis to the target cell. Granules
containing perforin and granzymes contribute to target
cell damage.
■ Ligation of Fas or the type-1 tumour necrosis factor
(TNF) receptor on the target cell leads to the activation
of caspases. The caspases are the ultimate mediators of
apoptosis in the target.
■ Myeloid cells induce damage in targets principally by
the release of toxic molecules. This is a reflection of
their normal function of killing pathogens.
Recognition of target cells by TC cells and NK cells
Fig. 10.1 Cytotoxic T cells recognize processed antigen
presented on the target cell by MHC molecules using their T-cell
receptor (TCR). Most TC cells are CD8+ and recognize antigen
presented by MHC class I, but a minority are CD4+ and recognize
antigen presented by MHC class II. By contrast, NK cells have
receptors that recognize MHC class I on the target and signal
inhibition of cytotoxicity. They use a number of different receptors
(NK receptors) to identify their targets positively for killing),
including CD2, CD69, or antibody bound to their Fc receptor (CD16).
are summarized briefly here. Cellular molecules that have
been partly degraded by proteasomes are transported to
the endoplasmic reticulum to become associated with
MHC class I molecules and are then transported to the cell
surface. Thus each cell samples its own molecules and
presents them for review by CD8+ TC cells. Both the cell’s
own molecules and those of intracellular pathogens will be
presented in this way.
Additional interactions may be required to stabilize the
bond between the TC cell and the target (Fig. 10.2), and
can even help to trigger the killing event. For example, by
adding antibodies against CD3 or CD2 on the Tc cell in
vitro, it is possible to trigger the killing of target cells that
are bound to the TC cell. It is probable that binding of
physiological ligands to these molecules can also trigger
TC cells in this way.
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CELL-MEDIATED CYTOTOXICITY
Interactions between Tc cells and target cells
Fig. 10.2 Some of the ligands involved in the interaction
between cytotoxic T cells and their targets.
Several viruses (particularly herpes viruses) have evolved
mechanisms to avoid recognition by Tc cells. They reduce
the expression of MHC molecules or even produce proteins which divert MHC molecules out of the endoplasmic
reticulum. This reduces the likelihood that processed viral
peptides will be presented at the cell surface. Because NK
cells specifically recognize cells which have lost their MHC
class I molecules, we can see that Tc cells and NK cells
act in a complementary way to protect the body. In effect,
the NK cells check that cells of the body are carrying their
identity card (MHC class I) while the Tc cell checks the
specific identity (antigen specificity) on that card.
Experiments involving the micromanipulation of individual cells have shown that a single cytotoxic T cell can
kill several target cells sequentially. To function in this
manner, Tc must be resistant to their own killing mechanisms and able to detach effectively from dying target cells.
Cytokine activated killer (LAK) cells are related
to NK cells
Immunologists have experimented with several potential
treatments for cancer. One approach has been to activate
the patient’s own lymphocytes in vitro with IL-2, and then
to reinfuse them. These cells, which are initially derived
from blood or spleen, are called cytokine activated killer
cells or LAKs. Such cells show enhanced MHC nonrestricted cytotoxicity, and they appear to be largely derived
from precursors that are indistinguishable from NK
cells. Thus LAK cells probably do not represent a separate
lineage, but rather a consequence of activation. This type
of cell is undergoing trials for the treatment of cancer in
humans.
NATURAL KILLER CELL RECEPTORS
NK cells are mostly derived from ‘large granular lymphocytes’ (LGLs), which comprise about 5% of human peripheral blood lymphocytes. The majority of NK cells are
CD3–CD16+CD56+CD94+ (see Appendix 2), and do not
164
contain productive rearrangements of the T-cell receptor
genes. Initial experiments on the specificity of NK cells
showed that MHC class I expression protected cells from
NK-cell mediated cytotoxicity and that particular allotypes
of HLA-C were dominant genes for producing resistance.
This lead to a search for receptors on NK cells which could
inhibit cytotoxicity and which might be expressed in a
variety of different forms, capable of reacting with MHC
class I and signalling its presence to the NK cell. Two
major types of molecules were identified and termed ‘killer
inhibitory receptors’ or KIRs. One group of molecules was
identified as type-2 membrane glycoproteins (C-terminus
outside) with a C-type lectin domain (Ca2+-dependent).
The other group of molecules were members of the Ig
superfamily. Subsequently, it was realized that while some
members of each group did indeed act as killer inhibitory
receptors, others could actually activate the killer cell. So,
it was not correct to call all members of both groups ‘inhibitory receptors’. Therefore it was proposed that the term
‘KIR’ now meaning ‘killer immunoglobulin-like receptor’,
should be used for just the second group of molecules.
The lectin-like receptor CD94 interacts with
HLA-E
The lectin-like receptor CD94 is a characteristic marker of
human NK cells, but is also found on a subset of Tc cells.
It covalently assembles with different members of another
group of type-2 membrane molecules called NKG2, and
the dimers are expressed on the cell membrane (Fig. 10.3).
There are at least six members of the NKG2 family
(NKG2A–NKG2F). The dimer of CD94-NKG2A is an
inhibitory receptor, which blocks NK cell-mediated cytotoxicity. By contrast, CD94-NKG2C is an activating
receptor. Although these two molecules are very similar,
they differ in their intracellular segments, and this determines whether the receptor is inhibitory or activating
(Fig. 10.3). In vivo, the role of the inhibitory version is
clear, but that of the activating variant is not. Possibly, it
may act as one of the receptors by which NK cells carrying
CD94-NKG2C actively engage their targets.
CD94 is distantly related to the mouse molecule Ly-49,
which is present in the NK cell gene complex (NKC).
Indeed, it was partly this homology of CD94 to mouse NK
cell receptors, that alerted researchers to the possibility
that CD94 was an NK cell receptor in man.
HLA-E presents peptides from other MHC class I
molecules
It is now known that the ligand for both CD94-NKG2A
and CD94-NKG2C is the HLA-E molecule. The HLA-E
gene locus encodes an MHC class I-like molecule. These
are sometimes called class 1b molecules, to distinguish
them from the classical MHC molecules which present
antigen to Tc cells. The extraordinary function of HLA-E
is to present peptides from other MHC class I molecules.
The leader peptides from other MHC molecules are
released in the endoplasmic reticulum, and these are required to stabilize functional HLA-E molecules (Fig. 10.4).
Cells lacking classical MHC class I molecules do not express HLA-E at the cell surface. Hence an inhibitory signal
is not passed to the NK cell. In effect HLA-E is a sensitive
Immunology 10
NATURAL KILLER CELL RECEPTORS
Lectin-like receptors of NK cells
mechanism for monitoring whether viruses or tumours
have downregulated MHC class I expression in a cell.
Although CD94 has a lectin domain, the recognition of
the HLA-E/peptide occurs via interaction with residues
from the peptide and the α1 and α2 domains of the MHC
molecule. The lectin domain could however reinforce this
interaction by binding to carbohydrate associated with the
MHC molecule.
KIRs are members of the immunoglobulin
superfamily
Fig. 10.3 The inhibitory receptors consist of the lectin-like CD94
disulphide bonded (red) to peptides from the NKG2 locus, such as
NKG2A which have intracellular domains carrying ITIM motifs
(immunoreceptor tyrosine inhibitory motif). The non-inhibitory
receptors, such as CD94/NKG2C, lack ITIMs, but have a charged
lysine (K) in the transmembrane segment which allows them to
interact with signal transducing molecules.
HLA-E presents peptides of other MHC class I molecules
The second group of NK cell receptors are members of the
immunoglobulin superfamily. They fall into two subsets,
having either two or three Ig domains (Fig. 10.5). A set of
approximately 12 such genes has been identified as a
cluster on chromosome 19q13.4. The two-domain members
(KIR2D) are defined as CD158, while the three-domain
members (KIR3D) were originally described as p70, based
on their molecular weights. Two of the two-domain
members have been shown to bind to allelic variants of
HLA-C, and these isoforms have inibitory cytoplasmic
domains (see below). The specificity of these receptors can
partly explain why particular HLA-C allotypes inhibited
some NK cells, as described above. It also explains why
these allotypes produce dominant resistance – if a cell
expresses a particular HLA-C allotype, then that is sufficient to inhibit a K cell which has engaged it.
Other isoforms of the KIRs have activating domains.
These generally engage HLA molecules with a lower affinity
than the inhibitory forms, and like the activating forms of
CD40-NKG2, their physiological functions are uncertain.
There is also a third group of receptors clustered in the
same region as the KIRs, which have either two or four
Killer immunoglobulin-like receptors
Fig. 10.4 Leader peptides from MHC class I molecules are loaded
onto HLA-E molecules in the endoplasmic reticulum, a process
which requires TAP transporters and tapasin to assemble
functional HLA-E molecules. These are presented at the cell
surface for review by the CD94 series of receptors on NK cells
(left). The MHC class I molecules meanwhile present antigenic
peptides from cytoplasmic proteins which have been transported
into the endoplasmic reticulum. These complexes are presented
to the TCR on CD8+ cytotoxic T cells.
Fig. 10.5 These receptors consist of either two or three
extracellular Ig superfamily domains. The inhibitory forms are
longer and have intracellular ITIMs, while the non-inhibitory forms
have the charged residue in the membrane comparable to the
non-inhibitory forms of CD94/NKG2.
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CELL-MEDIATED CYTOTOXICITY
Ig-like domains. They are called immunoglobulin-like
transcripts or ILTs and they have a wider cellular distribution than the other NK cell receptors, Some of these interact
with a broad spectrum of MHC molecules, and others with
none at all, hence their functions are still uncertain.
some controversial reports) polymorphs may also be active
against antibody-coated tumour targets. Some myeloid
cells (monocytes and eosinophils) are certainly important
effectors of damage to antibody-coated schistosomulae
(see Chapter 18).
HLA-G inhibits NK cell action against the
placenta
Intracellular signalling pathways coordinate
inhibitory and activating signals
An intriguing recent discovery is that the HLA-G molecule, which is expressed only on placental trophoblasts,
is a dominant NK-cell inhibitor that confers resistance to
all types of NK cells (HLA-G is another MHC class 1b
molecule). Trophoblast cells are derived from the fetus
and invade the maternal circulation as the placenta is
established. They are therefore allogeneic in the mother,
because they contain paternal MHC genes. However,
all conventional MHC genes are downregulated in these
cells, and the HLA-G expression is therefore required to
protect the placenta from attack by NK cells. There is some
debate as to which of the inhibitory receptors recognize
HLA-G. Both CD94 and several of the better-defined
KIRs have been excluded, but a likely candidate is ILT2.
The next question is how a NK cell decides between cytotoxic action or inaction. This decision is thought to depend
on the coordination of intracellular signalling pathways,
and may involve the balance between activating and
inhibitory signals. Both the lectin-like receptor and the
KIRs occur as inhibitory or activating molecules. The
key difference is the presence of ‘immunoreceptor tyrosine
inhibitory motifs’ or ITIMs. If these motifs become
phosphorylated, they can recruit phosphatases which
downregulate the activity of the NK cell (Fig. 10.7).
By contrast, other KIRs which lack the ITIMs can associate with a molecule (DAP12) which is related to the ζ
chain of the T-cell receptor complex. This molecule has
‘immunoreceptor tyrosine activation motifs’ (ITAMs),
which allow it to phosphorylate and recruit tyrosine kinases
including ZAP-70 (Fig. 10.7, and compare with figure 6.21),
which lead to cell activation. At present, it is not known
how the balance of activation and inhibition is resolved. In
particular it is uncertain how a ligand such as HLA-C will
act when it can bind to both inhibitory and activating
receptors on the same cell.
NK cells and K cells use several different
receptors to positively identify their targets
NK cells may engage their targets using a variety of receptors, including CD2, CD16, CD69 and receptors related
to those which inhibit cytotoxicity. The Fc receptor,
CD16, binds antibody bound to target cells and mediates
ADCC (Fig. 10.6). It is customary to refer to this as killer
(K) cell activity, but this function may also be performed
by several other cell types with Fc receptors, including T
cells. Myeloid cells expressing Fc receptors can also show
K-cell activity, but probably use killing mechanisms different
from those of T cells and NK cells (discussed later).
Potential targets for K-cell action include viral antigens
on cell surfaces, MHC molecules and some epitopes
present on tumours. Thus monocytes and (according to
Fig. 10.6 K-cell activity. Electron micrograph of a lymphocyte
(right) engaging a target cell sensitized with antibody (left). × 2500.
(Courtesy of Dr P. Penfold.)
166
Activation and inhibition by NK cell receptors
Fig. 10.7 Following phosphorylation of its ITIMs, the inhibitory
receptors of NK cells can bind to phosphatases, including SHP-1
and SHP-2, which inhibit killing. The non-inhibitory forms of the
receptor associate with a dimeric molecule DAP12, via the
complementary charged residues in their membanes. DAP12 has
activation motifs (ITAMs). When phosphorylated this recruits
kinases of the syk family or ZAP-70 (cf. T-cell activation,
Chapter 6). Whether this leads to NK cell activation, or whether it
modulates the inhibitory signals is not known.
Immunology 10
MECHANISMS OF CYTOTOXICITY
MECHANISMS OF CYTOTOXICITY
Cytotoxicity is effected by direct cellular
interactions, cytokines and granule exocytosis
Cytotoxic T cells, NK cells and K cells use a variety of
different mechanisms to kill their targets. These include
direct cell–cell signalling via surface molecules and indirect
signalling via cytokines. In addition, many CD8+ cytotoxic
T cells and large granular lymphocytes (NK cells and K
cells) have granules which contain proteins that damage
target cells if they are released directly against the target
cell plasma membrane. Exactly which combination of these
three mechanisms is used depends on the Tc cell involved.
Cytotoxic T cells and NK cells induce apoptosis
in their targets
Cells can die in two principle ways – by necrosis or apoptosis. Apoptosis is a highly ordered process in which cells
are systematically disassembled. The cells detach from their
neighbours and the cytoplasm and nucleus condense.
Mitochondria lose their membrane potential and leak cytochrome c into the cytoplasm. As the chromatin condenses,
it is cleaved into regular-sized fragments by endonucleases.
Finally the cell membrane starts to form blebs and the
cell may fragment into condensed apoptotic bodies. Dead
cells attract mononuclear phagocytes and are rapidly taken
up by phagocytosis to be broken down in phagolysomes.
Apoptosis is a feature of normal physiology. For example,
T cells which fail thymic selection die by apoptosis (see
Chapters 2, 12), as do B cells which are not selected during
affinity maturation in germinal centres (see Chapter 8).
This type of cell death also occurs extensively during organ
development, particularly in the central nervous system.
In each case the final stages of cell death involve the
activation of a group of enzymes called caspases, but the
events which trigger apoptosis are different for each
process.
By comparison necrosis is a less orderly event, in which
dying cells fall apart releasing their contents. This tends
to provoke macrophage activation and inflammation.
Another difference is that apoptosis requires energy (ATP)
while necrosis does not. The damage caused by pathogens
and the colateral damage caused by granulocytes and
macrophages acting against them induce cell death by
necrosis.
8-deficient mice are not susceptible to cell killing by the
mechanisms outlined below.
Cytotoxicity may be signalled via Fas or a TNF
receptor on a target cell
Cytotoxic T cells signal to their targets using members
of the TNF receptor group of molecules. These include
Fas (CD95) and the type 1 TNF receptor, TNFR-1, which
are widely distributed in the body (Fig. 10.8). Other members of the group are CD30 and CD40 which are involved
in lymphocyte differentiation The ligand for Fas (FasL)
is expressed on mature CD4+ and CD8+ T cells after
activation. Ligation of Fas induces trimerization of the
Fas molecules on the cell surface, which causes them to
associate with a transducing molecule which recruits and
activates caspases 8 or 10 (Fig. 10.9). Note that cell killing
mediated by Fas also occurs as part of the normal processes
of lymphocyte selection, during development. For cytotoxic lymphocytes which lack granules the Fas pathway is
thought to be the principle means of signalling to the
target.
Most CD8+ Tc cells, NK cells (and macrophages) have
vesicles containing TNF and lymphotoxin which can be
released onto a target cell. TNF acts in a very similar way
to the Fas ligand. It causes trimerization of the TNFR-1 so
that the receptor associates with adaptor proteins which
recruit caspases. Both TNFR-1 and Fas contain intracytoplasmic domains (death domains) which are found
on a number of proteins involved in cell survival. (Note
however that a different form of TNF receptor, TNFR-2,
lacks these intracytoplasmic segments and therefore does
not transduce signals for apoptosis.)
TNF receptor family molecules
Caspases mediate cell death by apoptosis
Caspases are a group of proteases which have the unusual
property of cleaving their substrates on the C-terminal side
of an aspartate residue. More than 10 caspases have been
identified in man. They are produced in a pro-enzyme
form and become activated by cleavage into two or three
subunits. The caspases have very wide-ranging effects within
a cell. They can affect cell structure, intracellular signalling,
cell cycle control, DNA integrity and repair, and intercellular adhesion. Studies with caspase gene knockout mice
have shown that different caspases are associated with particular forms of apoptosis. For example, caspase-3-deficient
mice die shortly after birth due to a failure of normal CNS
development, but the mechanisms of cell death induced by
cytotoxic cells are unaffected. Notably, however, caspase-
F i g .1 0 . 8 The molecule Fas (CD95), the two TNF receptors and the
lymphotoxin receptor are illustrated diagramatically. The extracellular domains are similar to those found in the NGF receptor.
Both Fas and TNFR-1 have death domains which are involved in
the recruitment of caspases. The ligands for these receptors are
indicated at the top. Lymphotoxin-α can form homotrimers or
heterotrimers with lymphotoxin-β. Up to 25 other members of
these families have been identified by data-base searching.
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10 Immunology
CELL-MEDIATED CYTOTOXICITY
Mechanisms of cell killing
Fig. 10.9 Ligation of CD95 or TNFR-1
causes trimerization of the receptors.
Death domains in the cytoplasmic
portion of CD95 bind to the adaptor
protein FADD (=MORT-1), which recruits
caspase 8 or 10. TNFR-1 can activate
either caspase 8 or 10, via TRADD and
FADD, or caspase 2 via RIP and RAIDD.
Caspase 8 can further activate other
caspases, and these in concert lead to
apoptosis of the target cell.
Activated caspase 8 can cleave and activate other caspases,
in addition to its own direct actions in the pathways of
apoptosis (Fig. 10.9).
Granules of cytotoxic T cells contain perforin
and granzymes
It was originally thought that all cytotoxicity was caused by
the release of granule proteins onto the target cell. Indeed
the processes described above were only identified when
it was realized that cells which lack granules could still
kill targets. The specific granules of NK cells and Tc cells
contain several proteins, including perforin and granzymes
(granule-associated enzymes). After binding to its target,
the Tc cell directs its granules towards the membrane
adjoining the target. Then, in a Ca2+-dependent phase,
the granule contents are discharged into the cleft between
the two cells. This process can be seen in time-lapse video
microscopy (Fig. 10.10).
Perforin is a monomeric pore-forming protein that is
related both structurally and functionally to the complement component, C9. The vesicles also contain a serine
esterase that may be involved in the assembly of the lytic
complex. In the presence of Ca 2+, the perforin monomers
bind to the target cell membrane and polymerize to form
transmembrane channels. Although in close contact with
the perforin, the Tc cell survives and can continue to kill
168
further targets. It is thought to be protected from autodestruction by a proteoglycan (chondroitin sulphate A)
which is also present in the vesicles, and which may bind
to and inactivate the perforin. Perforin-knockout mice have
Tc cells which display reduced but still functional cytotoxicity, implying that perforin cannot be the only mechanism
used by these cells.
Granzymes are a collection of serine esterases (enzymes)
which are also released upon granule exocytosis and
become active after release. They are not essential for cytotoxicity, as cells lacking granzymes may still be cytotoxic.
Some of the granzymes may interact with intracellular
pathways in the target cell to activate mechanisms which
trigger apoptosis and DNA degradation. In fact, it is notable
that granzyme B has the same unusual specificity as the
caspases (see above). In order to activate apoptosis pathways
in the target cell, the granzymes need to gain access to the
cytoplasm. It has been proposed that perforin and granzymes
act synergistically; the granzymes enter the target cell via
pores created by perforin. The ways in which granule proteins
can contribute to cytotoxicity are shown in Figure 10.11.
To summarize, CD8+ TC cells use both FasL and granule
release to kill their targets, CD4+ Tc cells use principally
FasL, and NK cells use primarily their granules. TNF may
contribute to the cytotoxic damage produced by any of
these cells.
Immunology 10
NON-LYMPHOID CYTOTOXIC EFFECTORS
Fig. 10.10 Intracellular reorganizations during effector–target cell
interaction. Early events in the interaction of Tc with specific
targets were studied with high-resolution cinematographic
techniques. Four frames (together with interpretative drawings) are
shown, taken at different times, of a Tc interacting with its target.
The location of the granules within the effector cell is indicated in
each case. Before contact with the target (a), the effector had
granules located in a uropod at the rear, and was seen to move
randomly by extending pseudopods from the organelle-free,
broad leading edge of the cell. Within 2 minutes of contacting the
target (b), the Tc had begun to round up and initiate granule
reorientation (c). After 10 minutes (d), the granules occupied a
position in the zone of contact with the target, where they appear
to be in the process of emptying their contents into the i
ntercellular space between the two cells. (Courtesy of Dr V.H.
Engelhard.)
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10 Immunology
CELL-MEDIATED CYTOTOXICITY
Granule-associated killing mechanisms
Fig. 10.11 The cytotoxic lymphoid cell
degranulates, releasing perforin and
various enzymes (granzymes) into the
immediate vicinity of the target cell
membrane. In the presence of Ca2+ there
is enzymic polymerization of the perforin
to form polyperforin channels on the
target cell (1). Enzymes which activate
the apoptosis pathways, degradative
enzymes or other toxic substances
released from the cytotoxic cell may pass
through the channels on the target and
cause cell damage or killing (2).
NON-LYMPHOID CYTOTOXIC EFFECTORS
Macrophages can damage targets using their
non-specific toxic effector systems or via
cytokines
A number of non-lymphoid cells may be cytotoxic to other
cells or invading microorganisms, such as bacteria or parasites. Cytotoxicity may be triggered specifically to a target
by antibody-dependent cell mediated cytotoxicity (ADCC)
or may involve a range of non-specific toxic mediators.
For example macrophages and neutrophils both express
FcγRI and FcγRII which allows them to engage tumours
by ADCC.
In general, macrophages and neutrophils aim to destroy
Mechanisms which may contribute to the cytotoxicity of
myeloid cells
pathogens by internalizing them and subjecting them to
toxic molecules and enzymes within the phagolysosome.
These include the production of reactive oxygen intermediates, toxic oxidants and NO detailed in Chapter 9, as well
as the secreted molecules such as neutrophil defensins,
lysosomal enzymes and cytostatic proteins. If the phagocyte fails to internalize its target, then these mediators
may be released into the extracellular environment and
contribute to localized cell damage. This action is referred
to as ‘frustrated phagocytosis’, and occurs when the target
is engaged by surface receptors, but is too large to phagocytose. The actions of the mediators produced by the phagocyte damage the target, rather than induce apoptosis. For
this reason, the cytotoxic action of these cells tends to
produce necrosis and inflammation.
Nevertheless, in the case of macrophages, activated
cells secrete TNF which can induce apoptosis in a similar
way to NK cells and Tc cells. For this reason, macrophages
can induce necrosis, apoptosis or a combination of both,
depending on the state of activation of the macrophages
and the target involved. The mechanisms by which myeloid
cells produce cytotoxic damage are shown in Figure 10.12.
Eosinophils mediate cytotoxicity by exocytosis
of their granules
Fig. 10.12 Reactive oxygen intermediates (ROIs) and reactive
nitrogen intermediates (RNIs), cationic proteins, hydrolytic
enzymes and complement proteins released from myeloid cells
may damage the target cell in addition to cytokine-mediated
attack.
170
Mature eosinophils are characterized by their granules,
which have a crystalloid core which binds the dye eosin.
Generally, eosinophils are only weakly phagocytic; they
ingest some bacteria following activation, but are less
efficient than neutrophils at intracellular killing. Their
major function appears to be the secretion of various toxic
granule constituents, following activation. They are therefore effective for the extracellular killing of microorganisms,
particularly large parasites such as schistosomes (see
Chapter 16).
Immunology 10
NON-LYMPHOID CYTOTOXIC EFFECTORS
The components of the eosinophil granule include
major basic protein (MBP), eosinophil peroxidase (EPO)
and eosinophil cationic protein (ECP). Major basic protein
(not to be confused with myelin basic protein of oligodendrocytes) is the major component of eosinophil
granules, forming the crystalloid core. It has been shown
to damage, and sometimes kill, parasites, but also damages
host tissue cells. Eosinophil peroxidase is a highly cationic
heterodimeric 71–77-kDa haemoprotein, which is distinct
from the myeloperoxidase of neutrophils and macrophages.
In the presence of H2O2, also produced by eosinophils,
EPO will oxidize a variety of substrates, including halide
ions to produce hypohalite. Indeed, this may represent
the eosinophils’ most potent killing mechanism for some
parasites. ECP is an eosinophil-specific toxin which is very
potent at killing many parasites, particularly the schistosomulae of Schistosoma mansoni. The molecule is a ribonuclease, which because of its high charge, binds avidly
to negatively charged surfaces. It is possible that it forms
membrane channels, which allow other mediators access to
the target organism. Other molecules produced by eosinophils are lysophospholipase and eosinophil-derived neurotoxin (EDN), which is also a ribonuclease but with strong
neurotoxic activity.
Degranulation of eosinophils can be triggered in a
CRITICAL THINKING
number of ways. Binding to IgG-coated parasites via surface Fc γRII triggers release of some mediators including
ECP, but not EPO. By contrast, triggering via FcεRII leads
to the release of EPO, but not ECP. Parasite killing may
involve contact-dependent degranulation or may simply
require deposition of toxins within the local tissue. Degranulation may also be triggered directly in vitro by several
cytokines, including IL-3, IL-5, granulocyte-macrophage–
colony-stimulating factor (GM-CSF), TNF, interferon-β
(IFNβ) and platelet activating factor (PAF). These mediators
also enhance ADCC-mediated degranulation. Eosinophils
are prominent in the inflammatory lesion of a number of
diseases, particularly atopic disorders of the gut, skin and
respiratory tract, where they are often closely associated
with fibrotic reactions. Examples are atopic exzema, asthma
and inflammatory bowel disease. Although eosinophils
may play some regulatory role in these conditions, such as
inactivating histamine, their toxic products and cytotoxic
mechanisms are a major cause of the tissue damage. For
example, in asthma, eosinophil granule proteins are detectable in the blood and lungs following asthmatic attacks.
MBP can kill some pneumocytes and tracheal epithelial
cells while EPO kills type II pneumocytes. MBP can also
induce mast cells to secrete histamine, thus exacerbating
allergic inflammation.
• Mechanisms of cytotoxicity
Lymphocytes from a normal individual were stimulated
in vitro by coculture with irradiated T lymphoma cells.
(Irradiation of these stimulator cells prevents them from
dividing in the coculture.) After 7 days the lymphocytes
were harvested and fractionated to obtain a population
of cytotoxic cells (CD8+), and a population of NK cells
(CD94+, CD16+). These effector cells were set up in a
cytotoxicity assay with the tumour cells as targets. The
tumour cells were labelled, in order to detect both DNA
fragmentation and cell lysis. The following results were
obtained in these assays.
Treatment
No effector cells
10.1 Why do the Tc cells lyse the targets and induce
DNA fragmentation? What are these cells
recognizing on the tumour cell surface?
10.2 Why might the NK cells cause some damage to the
tumour? Why does the presence of antibody to the
tumour enhance the cytotoxic capacity of the NK
cells?
10.3 Explain the result with purified perforin.
Lysis
DNA fragmentation
4%
1%
CD8 Tc cells
82%
80%
NK cells
12%
11%
5%
1%
NK cells + anti-tumour cell antibody
28%
28%
Purified perforin
95%
2%
+
Anti-tumour cell antibody
171
10 Immunology
CELL-MEDIATED CYTOTOXICITY
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